DESCRIPTION A preponderance of evidence suggests that neurons are a major source of Abeta in the brain. Little is known about the subcellular localization and processing of APP in central neurons, although it is known that APP undergoes both anterograde and retrograde transport. It is also known that APP is not found in appreciable amounts in neuronal plasma membranes. The fate of transported APP is therefore still a mystery. In preliminary studies the PI has been able to demonstrate that a majority of APP in synapses is found in a novel, rab5-rich, vesicular organelle. Rab5 is a small GTP-binding protein which is required for endocytosis in fibroblasts and is involved in the recycling of small synaptic vesicles in nerve terminals. The major focus of this application will be the further purification and characterization of this organelle, the determination of the role of rab5 (and other APP-associated proteins) in APP trafficking and processing, and the characterization of the novel organelle as a potential site of APP metabolism. Specific Aim 1: To localize APP within highly purified synaptosomes (containing nerve terminals and some postsynaptic elements), and purify and characterize the APP-containing synaptic organelles by conventional and/or immunological methods. Specific Aim 2: To determine the ability of specific proteins, associated with APP- containing synaptic organelles, to regulate the localization, trafficking, and processing of APP in cultured cells, including polarized epithelial cells and primary neuronal cultures. Specific Aim 3: To test the purified APP-containing synaptic organelles for their ability to metabolize APP and for the presence of proteases, including the a-secretase that the PI has recently identified, as well as for the presence of the Alzheimer-associated proteins, presenilin-1, presenilin-2, and apolipoprotein E.